Method of reducing metal ions to elemental metal in the vicinity of an oxido-reductase enzyme

Abstract

Disclosed are methods and materials for utilizing enzymes to act on metal ions in solution so that the ions are reduced to metal. Additionally disclosed is how to use enzymes to accumulate metal particles. The alteration of metal particles by enzymes interacting with the organic shell of the particles is also described. These methods enable a wide range of applications including sensitive detection of genes and proteins, use as probes for microscopy, nanofabrication, biosensors, and remediation.

1. A method of depositing elemental metal in the vicinity of an oxido-reductase enzyme, comprising:
 combining an oxido-reductase enzyme with metal ions, an oxidizing agent and a reducing agent; 
 incubating the oxido-reductase enzyme with the metal ions in the presence of the oxidizing agent and the reducing agent, whereby the metal ions are reduced to elemental metal; and 
 depositing the elemental metal in the vicinity of the oxido-reductase enzyme, wherein the metal ions are selected from the group consisting of silver, gold, iron, mercury, nickel, copper, lead, and cesium ions and a mixture thereof. 
 
     
     
       2. The method of  claim 1 , wherein the metal ions are silver ions. 
     
     
       3. The method of  claim 1 , wherein the metal ions are silver ions in a solution of silver acetate. 
     
     
       4. The method of  claim 1 , wherein the oxido-reductase enzyme is peroxidase. 
     
     
       5. The method of  claim 1 , wherein the oxido-reductase enzyme is horseradish peroxidase. 
     
     
       6. The method of  claim 1 , wherein the oxido-reductase enzyme is conjugated to streptavidin. 
     
     
       7. The method of  claim 1 , wherein the oxido-reductase enzyme is conjugated to an antibody. 
     
     
       8. The method of  claim 1 , wherein the oxidizing agent is an oxygen-containing oxidizing agent. 
     
     
       9. The method of  claim 1 , wherein the reducing agent is selected from the group consisting of hydroquinone, a hydroquinone derivative, and n-propyl gallate. 
     
     
       10. The method of  claim 1 , wherein the metal ions act as a substrate for the oxido-reductase enzyme. 
     
     
       11. The method of  claim 10 , wherein the oxido-reductase enzyme is peroxidase and the metal ions are incubated with the peroxidase in the presence of an organic substrate of the peroxidase. 
     
     
       12. The method of  claim 11 , wherein the organic substrate is 3,3′-diaminobenzidine or 5-bromo-4-chloro-3-indolyl phosphate. 
     
     
       13. The method of  claim 1 , wherein the step of incubating includes
 incubating the oxido-reductase enzyme with the metal ions: 
 adding the oxidizing agent and reducing agent to the mixture of the oxido-reductase enzyme and the metal ions; and 
 incubating the oxido-reductase enzyme with the metal ions in the presence of the oxidizing agent and the reducing agent. 
 
     
     
       14. The method of  claim 1 , wherein the step of incubating includes
 incubating the oxido-reductase enzyme with the metal ions; 
 adding the reducing agent to the mixture of the oxido-reductase enzyme and the metal ions; 
 adding the oxidizing agent to the mixture of the oxido-reductase enzyme. the metal ions and the reducing agent; and 
 incubating the oxido-reductase enzyme with the metal ions in the presence of the oxidizing agent and the reducing agent. 
 
     
     
       15. The method of  claim 1 , wherein the step of depositing includes depositing the elemental metal within about  1  micron of the oxido-reductase enzyme. 
     
     
       16. The method of  claim 1 , wherein the step of depositing includes depositing the elemental metal in the vicinity of the oxido-reductase enzyme within a cell. 
     
     
       17. The method of  claim 1 , further comprising: localizing the oxido-reductase enzyme in the area of a predetermined antigen. 
     
     
       18. The method of  claim 17 , wherein the predetermined antigen is a human cancer antigen. 
     
     
       19. The method of  claim 17 , wherein the predetermined antigen is a Her-2/neu protein. 
     
     
       20. The method of  claim 1 , further comprising: defining an antigen; and
 localizing the oxido-reductase enzyme to the area of the defined antigen. 
 
     
     
       21. The method of  claim 1 , further comprising: localizing the oxido-reductase enzyme in the area of a predetermined nucleic acid or nucleic acid probe. 
     
     
       22. The method of  claim 21 , wherein the predetermined nucleic acid is a Her-2/neu gene or a nucleic acid probe for a Her-2/ neu gene. 
     
     
       23. The method of  claim 1 , further comprising: defining a nucleic acid probe; and binding the oxido-reductase enzyme to the defined nucleic acid probe. 
     
     
       24. The method of  claim 1 , further comprising: binding the oxido-reductase enzyme to a member selected from the group consisting of antibody, antibody fragments, peptide, nucleic acids, nucleic acid probes carbohydrates, drugs, steroids, products from plants, animals, humans and bacteria, and synthetic molecules, where each member has an affinity for binding to particular targets. 
     
     
       25. The method of  claim 1 , further comprising: binding an antibody to a predetermined antigen in a tissue section; and binding the oxido-reductase enzyme to the antibody. 
     
     
       26. The method of  claim 25 , wherein the binding of the antibody to the oxido-reductase enzyme is through biotin-avidin interaction. 
     
     
       27. The method of  claim 26 , wherein the antibody is a biotinylated monoclonal antibody; and the oxido-reductase enzyme is conjugated with streptavidin. 
     
     
       28. The method of  claim 25 , wherein the tissue section is embedded in a solid support. 
     
     
       29. The method of  claim 28 , wherein the solid support is paraffin. 
     
     
       30. The method of  claim 1 , further comprising: binding a nucleic acid probe to a predetermined gene in the cells of a tissue section; and binding the oxido-reductase enzyme to the nucleic acid probe. 
     
     
       31. The method of  claim 30 , wherein the binding of the nucleic acid probe to the oxido-reductase enzyme is through biotin-avidin interaction. 
     
     
       32. The method of  claim 31 , wherein the nucleic acid probe is labeled with a fluorescent moiety; the oxido-reductase enzyme is conjugated to streptavidin; and the binding of oxido-reductase enzyme to the nucleic acid probe is through a biotinylated antibody against the fluorescent moiety. 
     
     
       33. The method of  claim 1 , wherein the metal ions are silver ion; and the method further comprises pretreating the oxido-reductase enzyme with gold ions prior to the step of incubating. 
     
     
       34. The method of  claim 33 , wherein the step of pretreating includes washing away residual gold ions prior to the step of incubating. 
     
     
       35. The method of  claim 1 , wherein the oxido-reductase enzyme is horseradish peroxidase; the metal ions are silver ion; the oxidizing agent is hydrogen peroxide; and the reducing agent is hydroquinone. 
     
     
       36. The method of  claim 1 , wherein the step of incubating includes incubating the oxido-reductase enzyme with the metal ions in the presence of the oxidizing agent and the reducing agent in a controlled pH buffer solution. 
     
     
       37. The method of  claim 36 , wherein the controlled pH buffer solution is a citrate buffer at about pH 3.8. 
     
     
       38. The method of  claim 1 , further comprising: stopping the deposition of the elemental metal to the vicinity of the oxido-reductase enzyme after a certain period of time. 
     
     
       39. The method of  claim 38 , wherein the step of stopping includes washing away residual metal ions from the oxido-reductase enzyme. 
     
     
       40. The method of  claim 1 , further comprising: detecting the elemental metal deposited in the vicinity of the oxido-reductase enzyme. 
     
     
       41. The method of  claim 1 , further comprising: detecting the elemental metal deposited in the vicinity of the oxido-reductase enzyme by autometallography. 
     
     
       42. The method of  claim 1  wherein the oxido-reductase enzyme is catalase. 
     
     
       43. The method of  claim 1  wherein the oxido-reductase enzyme is lactoperoxidase.